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The Development of Macrophage-Mediated Cell Therapy to Improve Skeletal Muscle Function after Injury.

Rybalko V, Hsieh PL, Merscham-Banda M, Suggs LJ, Farrar RP - PLoS ONE (2015)

Bottom Line: A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo.We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion.Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program.

View Article: PubMed Central - PubMed

Affiliation: Department of Kinesiology, The University of Texas at Austin, 1 University Station D3700, Austin, TX 78712, United States of America.

ABSTRACT
Skeletal muscle regeneration following acute injury is a multi-step process involving complex changes in tissue microenvironment. Macrophages (MPs) are one of the key cell types involved in orchestration and modulation of the repair process. Multiple studies highlight the essential role of MPs in the control of the myogenic program and inflammatory response during skeletal muscle regeneration. A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo. As such, MPs hold great promise for cell-based therapies in the field of regenerative medicine. In this study we used bone-marrow derived in vitro LPS/IFN-y-induced M1 MPs to enhance functional muscle recovery after tourniquet-induced ischemia/reperfusion injury (TK-I/R). We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion. Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program. Furthermore, we show that delivery of M1 (LPS/IFN-γ) MPs early in regeneration accelerates myofiber repair, decreases fibrotic tissue deposition and increases whole muscle IGF-I expression.

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Evan’s Blue dye staining of damaged myofibers at 4, 5, 6 and 7 days post reperfusion in muscles treated with saline or 2x106 M0 and M1 macrophages 24 h after TK-I/R injury.(A) Representative images of EBD+ staining (Day 4 (n = 3); Day 5 (n = 3); Day 6 (n = 1); Day 7 (n = 1)). Damaged fibers (EBD permeable) represented in red, DAPI (blue) used to counterstain cell nuclei; (B) Measurements of percent (%) fluorescent area. Days 4 and 5 post-I/R, n = 3/group, except D4-M0D1 n = 2; 3 fields of view/animal. Days 6–7, n = 2, 3 fields of view/animal. Values expressed as mean ± SEM. (*) p<0.05 compared to saline; (#) p<0.05 compared to M0D1; one-way ANOVA, Tukey-HSD post-hoc.
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pone.0145550.g006: Evan’s Blue dye staining of damaged myofibers at 4, 5, 6 and 7 days post reperfusion in muscles treated with saline or 2x106 M0 and M1 macrophages 24 h after TK-I/R injury.(A) Representative images of EBD+ staining (Day 4 (n = 3); Day 5 (n = 3); Day 6 (n = 1); Day 7 (n = 1)). Damaged fibers (EBD permeable) represented in red, DAPI (blue) used to counterstain cell nuclei; (B) Measurements of percent (%) fluorescent area. Days 4 and 5 post-I/R, n = 3/group, except D4-M0D1 n = 2; 3 fields of view/animal. Days 6–7, n = 2, 3 fields of view/animal. Values expressed as mean ± SEM. (*) p<0.05 compared to saline; (#) p<0.05 compared to M0D1; one-way ANOVA, Tukey-HSD post-hoc.

Mentions: Debris clearance by pro-inflammatory monocytes/MPs in vivo has been deemed critical for tissue repair. The ablation of MPs results in persistence of necrotic tissue at the site of injury [14, 15, 18, 20]. We hypothesized that increasing numbers of M1 (LPS/IFN-γ) MPs at the site of TK-I/R injury early after acute insult could facilitate faster debris clearance by LPS/IFN-γ primed MPs. We used Evan’s blue dye (EBD) injection to label damaged muscle fibers in vivo. EBD+ muscle fibers were evident in all groups 4 days post-reperfusion without significant differences. However, starting at 5 days post-reperfusion M1 (LPS/IFN-γ) MP treated muscles showed significantly lower levels of EBD staining (Fig 6). Analysis of later time points supports this finding, with no EBD+ fibers in M1 (LPS/IFN-γ) treatment group evident on days 6 and 7 post-TK-I/R injury. These findings can be attributed to either increased clearance of necrotic debris by M1 (LPS/IFN-γ) MPs or accelerated M1 (LPS/IFN-γ) MP-mediated stimulation of myogenic repair processes [21, 28].


The Development of Macrophage-Mediated Cell Therapy to Improve Skeletal Muscle Function after Injury.

Rybalko V, Hsieh PL, Merscham-Banda M, Suggs LJ, Farrar RP - PLoS ONE (2015)

Evan’s Blue dye staining of damaged myofibers at 4, 5, 6 and 7 days post reperfusion in muscles treated with saline or 2x106 M0 and M1 macrophages 24 h after TK-I/R injury.(A) Representative images of EBD+ staining (Day 4 (n = 3); Day 5 (n = 3); Day 6 (n = 1); Day 7 (n = 1)). Damaged fibers (EBD permeable) represented in red, DAPI (blue) used to counterstain cell nuclei; (B) Measurements of percent (%) fluorescent area. Days 4 and 5 post-I/R, n = 3/group, except D4-M0D1 n = 2; 3 fields of view/animal. Days 6–7, n = 2, 3 fields of view/animal. Values expressed as mean ± SEM. (*) p<0.05 compared to saline; (#) p<0.05 compared to M0D1; one-way ANOVA, Tukey-HSD post-hoc.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696731&req=5

pone.0145550.g006: Evan’s Blue dye staining of damaged myofibers at 4, 5, 6 and 7 days post reperfusion in muscles treated with saline or 2x106 M0 and M1 macrophages 24 h after TK-I/R injury.(A) Representative images of EBD+ staining (Day 4 (n = 3); Day 5 (n = 3); Day 6 (n = 1); Day 7 (n = 1)). Damaged fibers (EBD permeable) represented in red, DAPI (blue) used to counterstain cell nuclei; (B) Measurements of percent (%) fluorescent area. Days 4 and 5 post-I/R, n = 3/group, except D4-M0D1 n = 2; 3 fields of view/animal. Days 6–7, n = 2, 3 fields of view/animal. Values expressed as mean ± SEM. (*) p<0.05 compared to saline; (#) p<0.05 compared to M0D1; one-way ANOVA, Tukey-HSD post-hoc.
Mentions: Debris clearance by pro-inflammatory monocytes/MPs in vivo has been deemed critical for tissue repair. The ablation of MPs results in persistence of necrotic tissue at the site of injury [14, 15, 18, 20]. We hypothesized that increasing numbers of M1 (LPS/IFN-γ) MPs at the site of TK-I/R injury early after acute insult could facilitate faster debris clearance by LPS/IFN-γ primed MPs. We used Evan’s blue dye (EBD) injection to label damaged muscle fibers in vivo. EBD+ muscle fibers were evident in all groups 4 days post-reperfusion without significant differences. However, starting at 5 days post-reperfusion M1 (LPS/IFN-γ) MP treated muscles showed significantly lower levels of EBD staining (Fig 6). Analysis of later time points supports this finding, with no EBD+ fibers in M1 (LPS/IFN-γ) treatment group evident on days 6 and 7 post-TK-I/R injury. These findings can be attributed to either increased clearance of necrotic debris by M1 (LPS/IFN-γ) MPs or accelerated M1 (LPS/IFN-γ) MP-mediated stimulation of myogenic repair processes [21, 28].

Bottom Line: A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo.We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion.Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program.

View Article: PubMed Central - PubMed

Affiliation: Department of Kinesiology, The University of Texas at Austin, 1 University Station D3700, Austin, TX 78712, United States of America.

ABSTRACT
Skeletal muscle regeneration following acute injury is a multi-step process involving complex changes in tissue microenvironment. Macrophages (MPs) are one of the key cell types involved in orchestration and modulation of the repair process. Multiple studies highlight the essential role of MPs in the control of the myogenic program and inflammatory response during skeletal muscle regeneration. A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo. As such, MPs hold great promise for cell-based therapies in the field of regenerative medicine. In this study we used bone-marrow derived in vitro LPS/IFN-y-induced M1 MPs to enhance functional muscle recovery after tourniquet-induced ischemia/reperfusion injury (TK-I/R). We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion. Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program. Furthermore, we show that delivery of M1 (LPS/IFN-γ) MPs early in regeneration accelerates myofiber repair, decreases fibrotic tissue deposition and increases whole muscle IGF-I expression.

Show MeSH
Related in: MedlinePlus